Histone deacetylases (HDACs) were first identified through their ability to deacetylate the epsilon amino group of specific lysine residues within the N-terminal tail regions of histones to promote transcriptional repression or gene silencing. Several deacetylase enzymes that have sequence homology to HDACs have more recently been shown to deacetylate non-histone protein targets in vivo such as the p53 tumor suppressor protein for DNA repair regulation and alpha-tubulin for maintenance of cell integrity, suggesting that these proteins have even broader function than transcriptional regulation. The HDAC proteins fall into three classes and employ two different catalytic mechanisms. Class I and II HDACs show considerable sequence homology within the catalytic domain and do not use a cofactor for catalysis. The class III HDACs belong to the Sir2 protein family and show primary sequence and structural divergence with the class I/II HDACs. In addition, the Sir2 proteins employ a novel catalytic mechanism, whereby protein deacetylation is accompanied by NAD+ hydrolysis generating a novel O-acetyl-ADP-ribose intermediate and nicotinamide. A particularly exciting area of HDAC research relates to their implicated role in human cancer, including the involvement of the human class I HDACs in acute myeloid leukemia and the class III HDACs in the regulation of the p53 tumor suppressor protein. Indeed, HDAC inhibitors are currently in clinical trails as anticancer agents and hydroxamic acid-based HDAC inhibitors, such as SAHA and TSA, have already shown promising activity against several different solid tumors at well-tolerated doses. Despite the important biological role of HDAC proteins and their involvement in human cancer, their mechanism for catalysis, mode of substrate-specific binding, and the biochemical consequence of HDAC deacetylation is poorly understood. This lack of mechanistic information stems from a paucity of structural information on these enzymes. The overall goal of this project is to elucidate the mechanism of HDAC function through a combined structure/function approach on a subset of biologically well-characterized HDAC model proteins. The Specific Aims of the proposal are to (1) Characterize the structure/function of the yeast Sir2 homologue, Hst2; (2) Characterize the structure/function of the bacterial Sir2 homologue, CobB; (3) Determine the crystal structure of archaeal Af1-Sir2 bound to its cognate archaeal chromatin protein substrate, Alba; (4) Determine the structure of the archaeal Af1-Sir2 substrate, Alba; (5) Determine the structure of the class I/II HDACs, human HDAC6 and yeast Hos3. Together, these studies will provide new molecular insights into the mode of catalysis and substrate-specific binding by HDACs, as well as the biochemical consequence of HDAC deacetylation. Moreover, these studies will provide a scaffold for the design of small molecule inhibitors for specific histone deacetylase enzymes that may have applications for the treatment of HDAC-mediated cancers.